In recent years, development of pharmaceuticals using a mediator, for example, hormone, or a physiologically active substance, for example, an antibody or an enzyme, as a drug has been actively conducted. When these substances are administered into the body, since they are subjected to phagocytosis by the reticuloendothelial system and excretion in kidney and eliminated from the body, circulation in blood is low and sufficient drug efficacy is hardly obtained. Therefore, attempts to improve the circulation of drug in blood have been made by modifying the drug with a water-soluble polymer such as a saccharide or polyethylene glycol (PEG). It is known that the modification with polyethylene glycol reduces the immunogenicity and antigenicity of the drug while maintaining the stability of the drug. In order to increase the uniformity of the drug (particularly, in order to suppress variations of the circulation of drug in blood, immunogenicity, drug efficacy and the like), it is necessary to use a PEG modifier having a small polydispersity (weight average molecular weight (Mw)/number average molecular weight (Mn)). Up to now, PEGylated interferon and PEGylated preparation, for example, PEGylated hrGCSF, come onto the market and the usefulness of PEGylated technology becomes clear.
In order to modify a physiologically active substance, for example, a protein or an enzyme, with polyethylene glycol, it is necessary to convert a terminal hydroxyl group of polyethylene glycol to a reactive functional group. In the conversion to a reactive functional group, the remaining of an unreacted hydroxyl group causes byproduct of a high molecular weight PEG impurity, a PEGylated preparation having a different bonding and the like. Therefore, it is preferred that the conversion rate from a hydroxyl group to the reactive functional group is high.
It has particularly high usefulness to convert a terminal hydroxyl group of polyethylene glycol to a terminal carboxyl group. For example, the polyethylene glycol having a terminal carboxyl group can be converted to a succinimidyl ester, which is an active ester, by using a condensing agent, for example, N-hydroxysuccinimide and dicyclohexylcarbodiimide. Since the succinimidyl ester reacts with a primary amine under near physiological conditions to from a stable amide, it is most used as an acylating agent for protein modification. Of the succinimidyl esters, succinimidyl propionate and succinimidyl butanoate are relatively stable in an aqueous solution while exhibiting an appropriate reactivity to an amino group of protein or a physiologically active substance so that they are particularly useful for the modification of physiologically active substance.
As to the conversion of terminal hydroxyl group of polyethylene glycol to a terminal carboxyl group, several reports have heretofore been made. For example, in Patent Document 1 (particularly, Claim 1 thereof), a method of producing a carboxylic acid including allowing to react a polyhydric alcohol represented by formula: Q(OH)x+y with a carboxylic acid represented by formula: CH(R′)═C(R″)—COOR′″, and then hydrolyzing the resulting product is described (definitions in the formulae are same as those described in Patent Document 1).
Further, in Patent Document 2 (particularly, Claim 1 thereof), a method of producing a water-soluble non-peptidic polymer having a carboxyl group including allowing to react a water-soluble non-peptidic polymer having at least one hydroxyl group with a tertiary alkyl acrylate to form an ester of the polymer and then treating the ester of the polymer with a strong acid is described.
However, in the production methods described in Patent Documents 1 and 2, there is a problem in that a conversion rate of a hydroxyl group to a carboxyl group is less than 85% and is not so high. Specifically, in the examples of Document 1, the conversion rate is from 31.9 to 84.7%, and in the examples of Document 2, the conversion rate is from 69.1 to 73.8%. In the case where purity of pharmaceutical grade is required, it is necessary to separate a polymer having a terminal carboxyl group as the product from a polymer having a terminal hydroxyl group as the unreacted substance by ion exchange or reverse phase HPLC. Moreover, the production methods described in Patent Documents 1 and 2 have a problem in that the reactions require a long period of time.
In Patent Document 3 (particularly, Claim 19 thereof), a method of producing a carboxylic acid of water-soluble polymer including (a) step of allowing to react a water-soluble polymer segment having at least one alkoxide ion or thiolate ion with an ortho ester having a suitable leaving group to form an ortho ester of the water-soluble polymer, and (b) step of being subjected the ortho ester of the water-soluble polymer formed in step (a) to at least one hydrolysis step to obtain a carboxylic acid of the corresponding water-soluble polymer is described. However, in the production method described in Patent Document 3, there is a problem in that an expensive reagent, for example, trimethyl 4-bromoorthobutyrate, is used. Moreover, there is a problem in that in the synthesis of the ortho ester used in the production method described in Patent Document 3 a multi-stage step is required.
Furthermore, as a method of forming a terminal carboxyl group, oxidation of a terminal hydroxyl group (that is, oxidation of an alcohol) is exemplified. As a method of producing a carboxylic acid by oxidizing an alcohol, a method using a strong acid or a heavy metal compound (for example, chromium compound) is known. However, in Jones oxidation which is known as a typical oxidation method of an alcohol, a highly toxic chromium compound is used. Therefore, in the case where a compound used for pharmaceutical is produced by Jones oxidation in order to remove the chromium compound, there is a problem in that purification with high precision is required.